We have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], and have established that hypusine biosynthesis occurs by two sequential enzymatic reactions, deoxyhypusine synthesis and deoxyhypusine hydroxylation. Its synthesis in the eIF5A precursor at a single lysine residue represents the most specific post-translational modification known to date. Hypusine is essential for the activity of eIF-5A and for eukaryotic cell proliferation. Inhibitors of hypusine biosynthesis cause arrest in cell proliferation. Deoxyhypusine synthase catalyzes the transfer of the butylamine moiety of the polyamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form deoxyhypusine. We have purified this enzyme from rat testis, identified its gene in the yeast Saccharomyces cerevisiae and cloned the human cDNA. Inactivation of the deoxyhypusine synthase gene in yeast causes loss of cell viability providing direct evidence that the hypusine modification is vital for growth of yeast cells. We have characterized the physical and catalytic properties and studied the reaction mechanism of the enzyme. We have shown that deoxyhypusine synthesis occurs by way of four steps: i) NAD-dependent dehydrogenation of spermidine, ii) transfer of the butyl amine moiety from dehydrospermidine to a specific lysine of the enzyme to form an enzyme-imine intermediate, iii) transfer of the butylamine moiety from the enzyme intermediate to the eIF5A precursor, iv) reduction of the eIF5A imine intermediate to the deoxyhypusine-containing form. The active site residue that is involved in enzyme-intermediate formation has been identified asLys-329 for the human enzyme and Lys-350 for the yeast enzyme; thus, the mechanism of deoxyhypusine synthesis appears to be conserved in eukaryotes. The X-ray crystal structure of human deoxyhypusine synthase in a complex with NAD reveals NAD binding sites and an active site pocket where spermidine is presumed to bind. The role of a number of amino acids predicted to be involved in the binding of NAD, of spermidine, and those critical for the catalysis, was assessed by site-directed mutagenesis. Molecular modeling of the spermidine binding site should aid development of specific inhibitors of deoxyhypusine synthase that may be useful as anti-proliferative agents.
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